Zebrafish as a Model Organism for Immunology
The zebrafish'', Danio rerio'', has been used as a model organism since the 1930's. It started out as a model for development due to the visual clarity of embryos and larvae. Embryo development happens quickly and because of their transparent nature one can visualize cell biological events rather easily. This is useful when trying to make in vivo ''observations. The 1980's brought about the creation of some genetic mutants, cloning and other genetic approaches to enable researchers to gain a better understanding of vertebrate development through the zebrafish. It is now a staple organism in studying developmental biology with the creation of numerous developmental mutants and the sequencing of the zebrafish genome. Due to their genetic tractability there is increased interest in using zebrafish in studying human disease. 1 Using the Zebrafish to Identify Genes Involved in Disease There are several methods that can be used to identify genetic mutations involved in human disease. First is the forward genetics approach. This done by creating random mutants using ethylnitrosurea (ENU) or retroviruses to create a library of genetic mutants. Phenotypes can be easily visualized in the anatomy of the zebrafish due to their transparency enabling even whole organism time-lapse images of disease progression. To make identification of genes even easier, retroviral insertions can be tagged with green fluorescent protein to easily identify their location. Complex, multi-gene diseases can be studied using a single genetic mutant for a certain disease and identifying enhancers and inhibitors of disease progression. 1 The reverse genetics approach, where a specific genetic mutation is created and the phenotype analyzed, is another method zebrafish geneticists use to identify genes involved in human ailments. In zebrafish this is generally done with a method called TILLING, or targeting induced local lesions in genomes. TILLING is done by using a chemical mutagen called ethyl methanesulfonate and is combined with a method for identifying point mutations using PCR and the formation of heteroduplexes where there is a mismatch. Reverse genetics can also be done using retroviral insertions and deletions, or transiently using siRNAs. Transgenic fish can also be made using transposons, although traditional methods used in mice such as homologous recombination are not yet possible. 1,2 Zebrafish as a Model for Immunology Research Several features make zebrafish a good candidate as a model system to study immunology. Lymphoid development can be visualized much earlier than other organisms. Fertilization and development happen ''ex vivo allowing relatively easy tracking of development. Single site mutations and screening for mutant phenotypes can be achieved more quickly and easily and in a more cost-effective manner. Zebrafish are small and can be housed together in a small space. Chemical screens can be done by simply adding the compound to the water. Most human innate and adaptive immune response genes have counterparts in the zebrafish. In adult fish the kidney is homologous to the bone marrow as a site for hematopoesis. T cell development develops similarly in the thymus as in mammals. Progenitor T cells develop in the kidney and migrate to the thymus for further development. T cell developmental genes are similar to other mammals. The TCR-alpha locus is similar to other mammalian systems and variable elements undergo VDJ recombination as in other systems. MHC Class I-III have been identified in zebrafish and antigen presenting cells present antigen to T cell receptors in the thymus. B cells in zebrafish have quite a few noticeable differences. They only share the IgD and IgM antibody isotypes. They do not undergo class switch recombination and experience inefficient somatic hypermutation. Zebrafish are also without lymph nodes, Peyer's patches and splenic germinal centers. Toll-like receptors in zebrafish show a high degree of homology to mammalian systems. They also have a developed complement system. Zebrafish provide a great model for early infection with the use of fluorescently labeled bacteria or immune cells and the translucent nature of the fish. The main caveat to this is normal fish pathogens are maintained at 28 C and human pathogens are maintained at 37 C. Adaptive immunity models for zebrafish have been less established, with no current model for autoimmunity or general lymphoproliferative disease (as of 2008). Immune deficiency models have been made with the use of phenotype-driven screens. T cell proliferation models have been made with the c-myc and Notch1 oncogenes and one B cell proliferation model with the fusion TEL-AML1. 3,4 The Use of Zebrafish as a Model for Immunology in the Literature A study in 2012 used zebrafish a proof of concept model for bacterial meningitis using Streptococcus agalactiae ''as the infectious organism. The authors were able to cause disease in fish using both intraperitoneal and intramuscular infection and found mortality correlated with infection in a dose dependent manner. They also showed that at lower doses of infection the zebrafish were able to clear infection indicating that the immune system could functionally clear infection. The authors showed that the bacteria was able to cross the blood brain barrier as there was obvious swelling in the brain cavity post infection. Bacteria was also detected in the blood and brain post infection. Proinflammatory cytokine levels were measured post infection and steadily increased in the hours post infection. The authors then infected the fish with bacterial strains lacking various virulence factors to test whether these virulence factors affect infection outcome and if these strains lack the ability to cross the blood-brain-barrier. Infection with these strains showed greater survival rates and approximately 80% of fish showed no signs of bacterial infection in the brain. 5 References 1. Lieschke, G.J. et.al. ''Nature Reviews Genetics ''8, 353-367 (May 2007). doi:10, 1038/nrg2091. Animal Models of Human Disease: Zebrafish Swim into View. http://www.nature.com/nrg/journal/v8/n5/full/nrg2091.html 2. http://en.wikipedia.org/wiki/TILLING_(molecular_biology) 3. Yoder, Jeffrey, et. al. ''Microbes and Infection 4, Issue 14 (November 2002) 1469-1478. Zebrafish as an Immunological Model System http://www.sciencedirect.com/science/article/pii/S1286457902000291 4. Meeker, Nathan D. et. al. ''Immunology and Zebrafish: Spawning New Models of Human Disease. Developmental and Comparative Immunology. Volume 32, Issue 7, 2008. pp 745-757. 5. Patterson, Hayley et. al. ''Developmental and Comparative Immunology 38, Issue 3 (November 2012) 447-455. Adult zebrafish model of bacterial meningitis in ''Streptococcus agalactiae ''infection. http://www.sciencedirect.com/science/article/pii/S0145305X1200167X